The chemotactic behavior of E. coli provides the best-studied and most tractable system for elucidating the molecular machinery that also underlies more complex bacterial behaviors, such as host invasion and pathogenesis. The long-term objective of this work is to elucidate the molecular signaling mechanisms of the chemoreceptors that mediate chemotactic behavior in E. coli -- how these transmembrane molecules detect and amplify minute chemical signals in the cell's environment and how they generate and modulate intracellular signals to control the cell's flagellar motors. Although chemotaxis in E. coli is the best understood biological signaling system, the extraordinary detection sensitivity of bacterial chemoreceptors cannot be explained by known molecular mechanisms. Physical clustering of the cell's receptors may provide an important source of signal gain in the system. Recent genetic and crosslinking studies of the serine (Tsr) and aspartate (Tar) receptors of E. coli demonstrated that bacterial chemoreceptors may signal collaboratively in teams comprised of trimers of receptor dimers. Moreover, receptor teams can contain chemoreceptors with different detection specificities working together to control shared kinase molecules (CheA). The specific experimental aims of the proposed project address predictions of the receptor team hypothesis and the mechanisms of receptor signaling: (i) The relationship between team formation, cellular clustering, and signaling sensitivity of chemoreceptors will be investigated with Tsr trimer contact mutants, using fluorescent reporters and in vivo crosslinking to monitor macroscopic and molecular interactions between receptor molecules. If clustering contributes to signal gain, receptors that signal well should cluster well, whereas receptors that cluster poorly should signal poorly. (ii) Physical and functional interactions between chemoreceptors of different specificity will be investigated with Tsr mutants that block Tar function (epistasis) or that regain function in the presence of Tar (functional rescue). Tar and Tsr mutants with compensatory signaling defects will be isolated to examine the behavior of mixed receptor teams. (iii) The signaling properties and structural features of receptor teams will be explored with in vitro assays, using mutant receptors to control team composition and covalent dimers of receptor signaling fragments to control team geometry. (iv) Two new experimental tools will be developed to facilitate these studies: a detergent-solubilized system for studying native chemoreceptor signaling complexes; and chemoreceptor molecules that function with covalenfly joined subunits. These studies will provide important new perspectives on the issue of receptor clustering, a hallmark of biological signaling systems at every complexity level. [unreadable] [unreadable] [unreadable]